Induction of T-cell apoptosis by human herpesvirus 6.

The mechanisms of cell death in CD4+ T cells mediated by human herpesvirus 6 (HHV-6) were investigated. The frequency of cell death in the human CD4+ T-cell line JJHAN, which had been inoculated with HHV-6 variant A or B, appeared to be augmented by tumor necrosis factor alpha (TNF-alpha). Agarose gel electrophoresis of DNA from HHV-6-inoculated cells showed DNA fragmentation in multiples of the oligonucleosome length unit. The degree of DNA fragmentation increased when HHV-6-inoculated cells were cultured in the presence of TNF-alpha. Flow cytometry and Scatchard analysis of TNF receptors revealed an increase in the number of the p55 form of TNF receptors on JJHAN cells after HHV-6 inoculation. It also appeared that treatment with anti-Fas monoclonal antibody (MAb) induced marked apoptosis in HHV-6-inoculated cells. Transmission electron microscopy showed characteristics of apoptosis, such as chromatin condensation and fragmentation of nuclei, but virus particles were hardly detected in apoptotic cells. Two-color flow cytometric analysis using anti-HHV-6 MAb and propidium iodide revealed that DNA fragmentation was present predominantly in uninfected cells but not in productively HHV-6-infected cells. In addition, JJHAN cells incubated with UV light-irradiated and ultracentrifuged culture supernatant of HHV-6-infected cells appeared to undergo apoptosis. The present study demonstrated that both HHV-6 variants A and B induce apoptosis in CD4+ T cells by indirect mechanisms, as reported recently in human immunodeficiency virus type 1 infection.     

UV-B Irradiated Cell Lines Execute Programmed Cell Death in Various Forms

We investigated changes typical for apoptosis in various cell lines after UV-B irradiation. Using established methods for detection of apoptosis we demonstrate changes of cellular morphology, phosphatidylserine (PS) exposure, ollgonucleosomal DNA fragmentation and generation of hypochrome nuclei. To isolated high-molecular-weight (hmwt) DNA fragments we engaged a new method avoiding pulse field gel electrophoresis. Most UV-B irradiated cell lines showed oligonucleosomal DNA fragmentation, hypochrome nuclei, morphological changes, annexin-V binding and positive TUNEL reaction. However, no oligonucleosomal DNA fragmentation could be detected in Raji and HaCaT cells. Whereas HaCaT cells displayed all other changes typical for apoptosis, Raji cells were TUNEL negative, formed low amounts of hmwt DNA and showed an 'atypically' low hypochrome shift. Nevertheless, UV-B irradiated Raji cells excluded propidium iodide (PI), bound annexin-V and stopped proliferation. This suggests that Raji cells underwent growth arrest with exposure of PS being the only feature of apoptosis. However, in the presence of phagocytes expressing the phosphatidylserine receptor these cells would share the removal pathway with apoptotic cells. Since UV-B induced programmed cell death differs in dependence of cells under investigation, the failure to detect oligonucleosomal DNA fragmentation or chromatin condensation is not suitable to exclude programmed (apoptotic?) cell death.     

Tissue transglutaminase in the small intestine of the mouse as a marker for apoptotic cells. Colocalization with DNA fragmentation

Besides the morphological changes in cells undergoing apoptosis, such as chromatin condensation and cell shrinkage, histological demonstration of DNA fragmentation by in situ end labeling (ISEL) has been widely used for the demonstration of apoptotic cells in tissue sections. Although DNA fragmentation can be demonstrated in apoptotic cells and apoptotic bodies in most cases, there is no clear correlation of ISEL staining with apoptosis. It has often been demonstrated that, in many morphologically intact cells, nuclei with fragmented DNA can be found. Thus staining with ISEL for the detection of apoptosis is useful only in connection with other markers for apoptosis as, for example, characteristic morphological changes. Here we show that tissue transglutaminase protein is unequivocally expressed in apoptotic enterocytes as shown by DNA fragmentation and morphology. Tissue transglutaminase is not expressed in enterocytes with healthy morphology, although DNA fragmentation can be demonstrated in these cells. Thus the immunohistochemical demonstration of tissue transglutaminase may serve as a simple marker for apoptotic epithelial cells in tissue sections.     

Identification of nuclei from apoptotic, necrotic, and viable lymphoid cells by using multiparameter flow cytometry.

BACKGROUND: Methods widely used to detect apoptosis do not allow us to easily distinguish between nuclei from viable or necrotic cells. Even if apoptosis and necrosis seem to occur as alternatives at the single cell level, they could be present simultaneously in a cell population much more frequently than expected. For this reason, attention was focused on attempting to recognize, by multiparameter flow cytometry, the characteristics of viable cells and of apoptotic or necrotic dead cells. METHODS: Apoptosis and necrosis were induced in vitro in murine thymocytes and lymphocytes from adult peripheral blood by using dexamethasone or prostaglandin E2 treatment and heat shock at 60 degrees C or hydrogen peroxide, respectively. Traditional methods, such as DNA gel electrophoresis and propidium iodide staining followed by single-fluorescence analysis or annexin-V-fluorescein isothiocyanate plus propidium iodide staining by using flow cytometry, were compared with a new method. This method consisted of combined light-scatter and red fluorescence analysis by flow cytometry after isolation of nuclei by hypotonic solution as well as high-dose detergent treatment and DNA staining with propidium iodide. RESULTS: Results showed that, although traditional methods such as DNA-gel electrophoresis and single-parameter fluorescence flow cytometry analysis were unable, as expected, to discriminate among viability, apoptosis, and necrosis, our new method has enabled us to easily identify nuclei from viable, apoptotic, and necrotic cells. Results obtained by using our method were comparable to those obtained by using two-color analysis of cells after propidium iodide/annexin V staining. CONCLUSIONS: A highly reproducible, inexpensive, rapid, and easily accessible method of analysis has been developed for simultaneously detecting apoptosis and necro sis.     

Sustained enhancement of Ca(2+) influx by glibenclamide induces apoptosis in RINm5F cells

Cytosolic Ca(2+) elevations are known to be involved in triggering apoptosis in many tissues, but the effect of sustained enhancement of Ca(2+) influx on apoptosis in beta cells remains unknown. We have found that the viability of RINm5F cells is decreased dose-dependently by continuous exposure to glibenclamide at concentrations from 10(-7) to 10(-4) M, and that this effect is partially ameliorated by pretreatment with cycloheximide. Electrophoresis of the cells exposed to glibenclamide revealed ladder-like fragmentation characteristic of apoptosis, and which also is suppressed by cycloheximide pretreatment. By using terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining, we detected increased DNA fragmentation in the nuclei of the cells exposed to glibenclamide, and staining with Hoechst 33342 and propidium iodide showed a dose-dependent increase in the number of cells with the chromatin condensation and fragmentation in their nuclei that is characteristic of apoptosis. The effects of glibenclamide on cell viability and apoptotic cell death were partially inhibited by treatment with Ca(2+) channel blocker, and by reducing the extracellular Ca(2+) concentration during glibenclamide exposure, suggesting that they may be derived from increased Ca(2+) influx. Furthermore, only the percentage of apoptotic cells, and not that of necrotic cells, increased with the increasing intracellular Ca(2+) concentration during glibenclamide exposure. In conclusion, we have demonstrated that the sustained enhancement of Ca(2+) influx caused by glibenclamide exposure can induce apoptotic cell death in a pure beta cell line.     

Dose- and time-dependent effects of TNFalpha and actinomycin D on cell death incidence and embryo growth in mouse blastocysts

This study was undertaken to obtain information about characteristics of different types of induced apoptosis in preimplantation embryos. Freshly isolated mouse blastocysts were cultured in vitro with the addition of two apoptotic inductors--TNFalpha and actinomycin D--at various doses and times. The average number of nuclei and the percentage of dead cells were evaluated in treated embryos. Classification of dead cells was based on morphological assessment of their nuclei evaluated by fluorescence microscopy, the detection of specific DNA degradation (TUNEL assay), the detection of active caspase-3 and cell viability assessed by propidium iodide staining. The addition of both apoptotic inductors into culture media significantly increased cell death incidence in blastocysts. Their effects were dose and time dependent. Lower concentrations of inductors increased cell death incidence, usually without affecting embryo growth after 24 h culture. Higher concentrations of inductors caused wider cell damage and also retarded embryo development. In all experiments, the negative effect of actinomycin D on blastomere survival and blastocyst growth was greater than the effect of TNFalpha. Furthermore, the addition of actinomycin D into culture media increased cell death incidence even after 6 h culture. Differences resulted probably from diverse specificity of apoptotic inductors. The majority of dead cells in treated blastocysts were of apoptotic origin. Morphological and biochemical features of apoptotic cell death induced by both TNFalpha and actinomycin D were similar and had homologous profile. In blastomeres, similarly to somatic cells, the biochemical pathways of induced apoptosis included activation of caspase-3 and internucleosomal DNA fragmentation.     

Degeneration of cryopreserved bovine oocytes via apoptosis during subsequent culture

Cryopreservation causes a significant proportion of bovine oocytes to undergo degeneration during subsequent culture. We investigated the degeneration mechanism of cryopreserved oocytes. In vitro matured bovine oocytes were vitrified by the open-pulled straw (OPS) method. In each replicate, a group of oocytes were randomly taken after warming to determine oocyte survival by both morphological evaluation and propidium iodide vital staining. The remainders were evaluated by morphological criterion. Morphologically intact oocytes were co-incubated with frozen-thawed spermatozoa for subsequent development. In situ examination of DNA breaks in oocytes and embryos was conducted using a Fluorescein-FragEL DNA fragmentation detection kit. A caspase-3 detection kit was used to detect caspase-3 activity in oocytes and embryos. Most of the oocytes survived cooling and warming processes as assessed by both morphological evaluation and vital stain. During subsequent culture, some degenerating oocytes displayed observable apoptotic morphology, such as cytoplasmic condensation, cytoplasmic fragmentation, and formation of apoptotic bodies. Biochemical markers of apoptosis, such as apoptotic DNA fragmentation and activation of caspases, were detected not only in oocytes having typical apoptotic morphology, but also in oocytes without observable apoptotic morphology. In embryos, positive signals for both biochemical markers were detected in blastomeres. This experiment suggests that cryopreserved bovine oocytes degenerate via apoptosis during subsequent culture.     

Apoptotic and non-apoptotic cell death in hormone-dependent glands

The proliferation of cells and cell death are involved in the maintenance of appropriate tissue homeostasis. In the present study, two different mechanisms of cell death were identified in the prostate and pituitary glands when morphological data, fragmentation of DNA, and TUNEL labelling of apoptotic nuclei were compared. Typical cell death by apoptosis was identified by morphological and molecular approaches in the prostate after orchidectomy. By contrast, neither DNA fragmentation nor TUNEL labelling were found in dead cells occurring in the pituitary gland after interruption of lactation. Regressing lactotrophs were characterised by condensation and disruption of the cytoplasmic matrix, but preserved intact nuclei until advanced stages of regression. Degenerating “dark” cells comparable to those described in the pituitary were also seen coexisting with typical apoptosis in the prostate epithelial lining of orchidectomised rats. Both forms of cell death could be clearly differentiated, because dark cells suffer severe alterations of cytoplasmic organelles while maintaining the integrity of the nucleus. In contrast, apoptotic cells present well-preserved cytoplasmic organelles, but grossly disrupted nuclei with fragmentation and condensation of chromatin.     

Role of liver sinusoidal endothelial cells and stabilins in elimination of oxidized low-density lipoproteins

Atherogenesis is associated with elevated levels of low-density lipoprotein (LDL) and its oxidized form (oxLDL) in the blood. The liver is an important scavenger organ for circulating oxLDLs. The present study aimed to examine endocytosis of mildly oxLDL (the major circulating form of oxLDLs) in liver sinusoidal endothelial cells (LSECs) and the involvement of the scavenger receptors stabilin-1 and stabilin-2 in this process. Freshly isolated LSECs, Kupffer cells (KCs), and stabilin-1- and stabilin-2-transfected human embryonic kidney cells were incubated with fluorescently labeled or radiolabeled oxLDLs [oxidized for 3 h (oxLDL(3)), 6 h, or 24 h (oxLDL(24))] to measure endocytosis. The intracellular localization of oxLDLs and stabilins in LSECs was examined by immunofluorescence and immunogold electron microscopy. Whereas oxLDL(24) was endocytosed both by LSECs and KCs, oxLDL(3) (mildly oxLDL) was taken up by LSECs only. The LSEC uptake of oxLDLs was significantly inhibited by the scavenger receptor ligand formaldehyde-treated serum albumin. Uptake of all modified LDLs was high in stabilin-1-transfected cells, whereas stabilin-2-transfected cells preferentially took up oxLDL(24), suggesting that stabilin-1 is a more important receptor for mildly oxLDLs than stabilin-2. Double immunogold labeling experiments in LSECs indicated interactions of stabilin-1 and stabilin-2 with oxLDL(3) on the cell surface, in coated pits, and endocytic vesicles. LSECs but not KCs endocytosed mildly oxLDL. Both stabilin-1 and stabilin-2 were involved in the LSEC endocytosis of oxLDLs, but experiments with stabilin-transfected cells pointed to stabilin-1 as the most important receptor for mildly oxLDL.     

Reactive oxygen species induce signals that lead to apoptotic DNA degradation in primary CD4+ T cells

Reactive oxygen species are toxic to cells but they may also have active roles in transducing apoptotic events. To study the role of reactive oxygen species in growth factor depletion induced apoptosis of human primary CD4+ T cells, we used a synthetic manganese porphyrin superoxide dismutase mimetic to detoxify superoxide anions formed during apoptosis. Apoptosis of primary CD4+ T cells was characterized by generation of superoxide anions, plasma membrane phosphatidyl-serine translocation, loss of mitochondrial membrane potential, activation of caspase 3, condensation of chromatin, as well as DNA degradation. The detoxification of superoxide anions did not influence plasma membrane phosphatidyl-serine translocation, or chromatin condensation, and only marginally inhibited the loss of mitochondrial membrane potential and the formation of DNA strand breaks. In contrast, the detoxification of superoxide anions significantly reduced caspase 3 activity and almost completely inhibited the apoptotic decrease in total cellular DNA content as measured by propidium iodide staining. Our results indicate that reactive oxygen anions induce signals leading to efficient DNA degradation after the initial formation of DNA strand breaks. Thus, reactive oxygen anions have active roles in signaling that lead to the apoptotic events.     

Supravital exposure to propidium iodide identifies apoptosis on adherent cells

BACKGROUND: Several studies indicate that plasma membrane changes during apoptosis are a general phenomenon. Among the flow cytometric methods to measure apoptosis, the Annexin V assay that detects the membrane exposure of phosphatidylserine (PS) is one of the most commonly used. However, the various treatments used for the detachment of adherent cells generally interfere with the binding of Annexin V to membrane PS, making apoptosis measurement a technical problem. Materials and Methods Apoptosis of different cell lines was investigated by fluorescence microscopy and multiple flow assays designed to assess loss of membrane integrity, translocation of PS, DNA fragmentation, and light scatter changes. Results and Conclusions We show that supravital propidium iodide (PI) assay stains adherent apoptotic cells, allowing flow cytometric quantification. Moreover, supravital exposure to PI without prior permeabilization identifies apoptotic cells as well as Annexin V and permits the simultaneous surface staining by FITC- and PE-conjugated monoclonal antibodies. As in the case of necrotic or permeabilized cells, fluorescence microscopy has revealed that PI staining of apoptotic cells is localized in the nucleus. This suggests that the binding of PI to the DNA/RNA structures is stable enough to withstand the trypsinization and/or washing procedures necessary to detach adherent cells.     

Induction of apoptosis in mouse thymocytes by microcolin A and its synthetic analog

Microcolin A (Mic-1), a marine-derived compound, has been shown to be a novel antiproliferative and immunosuppressive agent. We investigated the ability of Mic-1 and its chemosynthetic analog, microcolin A3 (Mic-3), to induce apoptosis in murine thymocytes. Following incubation of the cells with Mic-1 (10-100 nM) or Mic-3 (10-100 nM), internucleosomal DNA fragmentation in apoptotic cells was detected by agarose gel electrophoresis and the diphenylamine (DPA) assay; the presence of hypodiploid nuclei assessed by propidium iodide (PI) staining; and the percentages of apoptotic and necrotic cells quantified by morphological observation and fluorescein labeled annexin-V binding. Our results show that both Mic-1 and Mic-3 are potent inducers of apoptosis in thymocytes depending on drug concentration and time of exposure, with Mic-3 being more potent than Mic-1 in the induction of apoptosis. Furthermore, flow cytometric analysis using monoclonal antibodies specific to thymocyte subpopulations showed that the proportion of the early immature CD4+ CD8+ T-cell subpopulation in thymocytes was selectively decreased by both agents with a corresponding increase of other subpopulations, indicating that CD4+ CD8+ T cells are the most likely targets of Mic-1 and Mic-3. These in vitro results suggest that the antiproliferative and immunosuppressive properties of both compounds are possibly associated with apoptosis-inducing events and imply that they may have additional potential value as antineoplastic agents.     

Analysis of cycloheximide-induced apoptosis in human leukocytes: fluorescence microscopy using annexin V/propidium iodide versus acridin orange/ethidium bromide

Apoptosis is a highly regulated and programmed cell breakdown process characterized by numerous changes. Since it is implicated in many pathological as well as physiological processes, it is vital to have reliable methods for detecting cell death. In this study, we compared several methods for detecting apoptosis and necrosis in human leukocytes. Apoptosis was induced either by incubating the cells with various doses of cycloheximide (CHX) or by 312 nm UVB irradiation. The methods used for detecting apoptosis were light microscopy (May Grunwald-Giemsa and trypan blue staining), fluorescence microscopy (acridin orange/ethidium bromide and annexin V/propidium iodide staining) and agarose gel electrophoresis of fragmented genomic DNA. Our study showed that CHX-induced apoptosis in cultured peripheral blood mononuclear cells but had no effect on apoptosis in polymorphonuclear cells, so its effect depends on cell type. Evaluation and comparison of the methods for detecting apoptosis showed the following. A Giemsa-stained cytospin allows the main morphological characteristics of necrotic and apoptotic death to be recognized. Trypan blue staining, widely used for estimating cell viability, is valueless for detecting apoptosis. Both fluorescence methods provided reliable and reproducible results and distinguished clearly between subpopulations of apoptotic cells, and were closely intercorrelated. Although applicable to a wide spectrum of cell types, agar electrophoresis of extracted DNA cannot be applied to all cell types and apoptotic conditions. Generally, microscopic examination of acridin orange/ethidium bromide stained cells can be recommended as the most reliable of the methods tested.     

Aged mice exhibit distinct peripheral B-cell phenotypes differing in apoptotic susceptibility: an ex vivo analysis.

BACKGROUND: Age-related changes in the antibody response have been classically associated with alterations in T-cell help, but increasing evidence shows that intrinsic B-cell defects exist. This article analyzes the apoptotic susceptibility of peripheral B-cells in aged and young control mice. MATERIALS AND METHODS: Freshly isolated lymphocytes from spleen and Peyer's patches (PPs) were labeled for B-cell lineage (B220(+) cells) and germinal center B subset (GCs, B220(+)/PNA(+) cells). Alternatively, splenic B-cells purified by MACS were used. Apoptosis was monitored by the Annexin V binding, incorporation of 3,3(')-dihexyloxacarbocyanine iodide (DiOC(6)(3)), propidium iodide (PI) staining, and morphological changes. Moreover, intracellular Bcl-2 expression and Bad phosphorylation status were also analyzed in B-cells. RESULTS: We showed in aging mice an enhanced Annexin V(+)/PI(-) cell percentage in splenic B-lymphocytes, which was correlated with a lower DeltaPsi(m). By contrast, no change in apoptosis was observed in compartments known to be enriched in activated B-cells (GCs and PPs). Analysis of Bcl-2 levels revealed no modification. When using B-cells purified by MACS, we strongly confirm data obtained on staining cells. Moreover, enhanced spontaneous apoptosis of splenic B-cells in aged mice was found to be correlated with a reduced phosphorylated Bad expression. CONCLUSION: Increased apoptosis of resting B-cells in old mice may be determined by an altered Bad phosphorylation, which in turn contributes to cell death by lowering the mitochondrial threshold for apoptosis.     

Apoptotic nuclear morphological change without DNA fragmentation.

Apoptosis is characterized morphologically by condensation and fragmentation of nuclei and cells and biochemically by fragmentation of chromosomal DNA into nucleosomal units [1]. CAD, also known as CPAN or DFF-40, is a DNase that can be activated by caspases [2] [3] [4] [5] [6]. CAD is complexed with its inhibitor, ICAD, in growing, non-apoptotic cells [2] [7]. Caspases that are activated by apoptotic stimuli [8] cleave ICAD. CAD, thus released from ICAD, digests chromosomal DNA into nucleosomal units [2] [3]. Here, we examine whether nuclear morphological changes induced by apoptotic stimuli are caused by the degradation of chromosomal DNA. Human T-cell lymphoma Jurkat cells, as well as their transformants expressing caspase-resistant ICAD, were treated with staurosporine. The chromosomal DNA in Jurkat cells underwent fragmentation into nucleosomal units, which was preceded by large-scale chromatin fragmentation (50-200 kb). The chromosomal DNA in cells expressing caspase-resistant ICAD remained intact after treatment with staurosporine but their chromatin condensed as found in parental Jurkat cells. These results indicate that large-scale chromatin fragmentation and nucleosomal DNA fragmentation are caused by an ICAD-inhibitable DNase, most probably CAD, whereas chromatin condensation during apoptosis is controlled, at least in part, independently from the degradation of chromosomal DNA.     

Comparison of several techniques for the detection of apoptotic astrocytes in vitro

Implication of apoptosis in numerous physiological and pathological processes has resulted in the development of numerous methods to detect apoptosis, but none of them is adapted to all cell types. In this study, we induced apoptosis on murine immortalized astrocytes with urine from multiple sclerosis (MS) patients. Among techniques allowing the detection of apoptotic cells, only a few are adapted to adherent cells such as astrocytes. We compared several techniques (propidium iodide labelling and flow cytometry analysis, TUNEL and annexin V labelling in immunofluorescence, DNA ladder, ELISA tests to detect nucleosomes) in order to choose the method best adapted to our adherent cellular model and to discuss their practicability for the detection of apoptosis on adherent cells.
For technical course, propidium iodide labelling followed by flow cytometry analysis as a quantitative technique, and TUNEL in IF (easier and quicker than propidium iodide) as a semiquantitative test were both retained as best adapted to our case.
Moreover, in our model, we have observed that phosphatydilserine externalization and DNA fragmentation were concomittant after induction of apoptosis.
Techniques studied in this article would allow an enlarged study of the apoptotic mechanism in several pathologies by culture of adherent cells sensitive to apoptosis in vitro .     

Differential kinetics of propidium iodide uptake in apoptotic and necrotic thymocytes

Apoptosis and necrosis represent two different mechanisms by which cells die. The dynamics of cellular lesions in these two processes differ. In particular we demonstrate that plasma membrane damage, occurring as a primary event during necrosis represents, on the contrary, a delayed but massive phenomenon during apoptosis. In consequence there are different kinetics of propidium iodide incorporation by necrotic and apoptotic thymocytes. This represents the basis for the flow cytometric identification of different cellular subsets. Analysis of these subsets after sorting showed that clearly apoptotic cells, which are not able to exclude propidium iodide for long incubation periods, do not show any morphologically detectable membrane damage. The kinetics of propidium iodide incorporation in vivo in isolated rat thymocytes can therefore be used in flow cytometric analysis. This technique can be used instead of DNA staining of ethanol-treated cells or nick translation to recognize apoptotic cells, and distinguish apoptosis from necrosis, without killing the cell.     

Activation of caspase-3-dependent and -independent pathways during 7-ketocholesterol- and 7beta-hydroxycholesterol-induced cell death: a morphological and biochemical study

On treatment with 7-ketocholesterol (7-keto) or 7beta-hydroxycholesterol (7beta-OH), which are major oxysterols in atherosclerotic plaques, the simultaneous identification of oncotic and apoptotic cells suggests that these compounds activate different metabolic pathways leading to various modes of cell death. With U937, MCF-7 (caspase-3 deficient), MCF-7/c3 cells (stably transfected with caspase-3), we demonstrate that caspase-3 is essential for caspase-9, -7, -8 activation, for Bid degradation mediating mitochondrial cytochrome c release, for cleavage of poly(ADP-ribose) polymerase and inhibitor of the caspase-activated deoxyribonuclease, and, at least in part, for internucleosomal DNA fragmentation. The crucial role of caspase-3 was supported by the use of z-VAD-fmk and z-DEVD-fmk, which abolished apoptosis and the associated events. However, inactivation or lack of caspase-3 did not inhibit 7-keto- and 7beta-OH-induced cell death characterized by staining with propidium iodide, loss of mitochondrial potential. The mitochondrial release of apoptosis-inducing factor and endonuclease G was independent of the caspase-3 status, which conversely played major roles in the morphological aspects of dead cells. We conclude that caspase-3 is essential to trigger 7-keto- and 7beta-OH-induced apoptosis, that these oxysterols simultaneously activate caspase-3-dependent and/or -independent modes of cell death.     

Caspase-independent apoptotic pathways in T lymphocytes: a minireview

Cell death by apoptosis is involved in the maintenance of T cell receptor diversity, self tolerance, and T-cell number homeostasis. Until recently, apoptosis was thought to require caspase activation. Evidence is now accumulating that a caspase-independent pathway exists, shown by in vitro experiments with broad-range caspase inhibitors. Mature T lymphocytes readily undergo caspase-independent apoptosis in vitro, and recent data suggest that this type of apoptosis may be involved in the negative selection of thymocytes. Mitochondria likely release death triggers specific for both caspase-dependent and caspase-independent apoptotic pathways (cytochrome c and AIF respectively) in response to apoptotic stimuli. A caspase-independent pathway is triggered first in activated T lymphocytes subjected to apoptotic stimuli that do not rely on receptors with death domains. In this pathway, the early commitment phase to apoptosis involves cell shrinkage, peripheral DNA condensation and the translocation of mitochondrial AIF to the cytosol and nucleus. This process is reversible until mitochondrial cytochrome c is released and m dissipated. Only at this stage are caspases activated.     

Loss of Acinus inhibits oligonucleosomal DNA fragmentation but not chromatin condensation during apoptosis

Chromatin condensation and oligonucleosomal DNA fragmentation are the nuclear hallmarks of apoptosis. A proteolytic fragment of the apoptotic chromatin condensation inducer in the nucleus (Acinus), which is generated by caspase cleavage, has been implicated in mediating apoptotic chromatin condensation prior to DNA fragmentation. Acinus is also involved in mRNA splicing and a component of the apoptosis and splicing-associated protein (ASAP) complex. To study the role of Acinus for apoptotic nuclear alterations, we generated stable cell lines in which Acinus isoforms were knocked down by inducible and reversible RNA interference. We show that Acinus is not required for nuclear localization and interaction of the other ASAP subunits SAP18 and RNPS1; however, knockdown of Acinus leads to a reduction in cell growth. Most strikingly, down-regulation of Acinus did not inhibit apoptotic chromatin condensation either in intact cells or in a cell-free system. In contrast, although apoptosis proceeds rapidly, analysis of nuclear DNA from apoptotic Acinus knockdown cells shows inhibition of oligonucleosomal DNA fragmentation. Our results therefore suggest that Acinus is not involved in DNA condensation but rather point to a contribution of Acinus in internucleosomal DNA cleavage during programmed cell death.